EP1700865A1 - Photoproteine mit erhöhter Biolumineszenz und deren Verwendung als intrazelluläre Calcium-Indikatoren - Google Patents

Photoproteine mit erhöhter Biolumineszenz und deren Verwendung als intrazelluläre Calcium-Indikatoren Download PDF

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EP1700865A1
EP1700865A1 EP05005390A EP05005390A EP1700865A1 EP 1700865 A1 EP1700865 A1 EP 1700865A1 EP 05005390 A EP05005390 A EP 05005390A EP 05005390 A EP05005390 A EP 05005390A EP 1700865 A1 EP1700865 A1 EP 1700865A1
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photoprotein
seq
cell
calcium
bioluminescence
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French (fr)
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Nadia Mastroianni
Silvia Cainarca
Sabrina Corazza
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AXXAM Srl
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AXXAM Srl
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Priority to EP05005390A priority Critical patent/EP1700865A1/de
Priority to EP06723320A priority patent/EP1858921B1/de
Priority to ES06723320T priority patent/ES2321029T3/es
Priority to US10/587,523 priority patent/US7981602B2/en
Priority to CA002601029A priority patent/CA2601029A1/en
Priority to CNA200680007800XA priority patent/CN101137667A/zh
Priority to AU2006222182A priority patent/AU2006222182A1/en
Priority to PT06723320T priority patent/PT1858921E/pt
Priority to DE602006005141T priority patent/DE602006005141D1/de
Priority to RU2007133797/10A priority patent/RU2407750C2/ru
Priority to AT06723320T priority patent/ATE422504T1/de
Priority to PCT/EP2006/002172 priority patent/WO2006094805A1/en
Priority to KR1020077021664A priority patent/KR20070121689A/ko
Priority to JP2008500125A priority patent/JP4962981B2/ja
Priority to DK06723320T priority patent/DK1858921T3/da
Publication of EP1700865A1 publication Critical patent/EP1700865A1/de
Priority to IL185857A priority patent/IL185857A/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43595—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from coelenteratae, e.g. medusae
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/07—Fusion polypeptide containing a localisation/targetting motif containing a mitochondrial localisation signal

Definitions

  • the present invention relates to photoproteins with enhanced bioluminescence and to their use as intracellular calcium indicators.
  • the photoproteins are obtained by mutagenesis of the clytin coding sequence and show enhanced bioluminescence, high affinity for calcium and long-lasting light emission. They are conveniently used in cell-based assays to determine variations of intracellular calcium concentration, particularly in assays for the screening of molecules with high and ultra-high-throughput techniques.
  • Bioluminescence is the phenomenon by which visible light is emitted by living organisms or by a substance derived from them through a variety of chemiluminescent reaction systems. Bioluminescence reactions require three major components: a luciferin, a luciferase and molecular oxygen. However, other components may also be required in some reactions, including cations (Ca ++ and Mg ++ ) and cofactors (ATP, NAD(P)H). Luciferases are enzymes that catalyse the oxidation of a substrate, luciferin, and produce an unstable intermediate. Light is emitted when the unstable intermediate decays to its ground state, generating oxyluciferin.
  • luciferin/luciferase system can be extracted in the form of a stable "photoprotein" which emits light upon calcium binding.
  • Photoproteins differ from luciferases in that they are stabilized oxygenated intermediate complexes of luciferase and luciferin.
  • Photoproteins are present in many marine coelenterates and allow these organisms to emit light for a variety of purposes including breeding, feeding and defense (1), There are many luminescent organisms, but only seven photoproteins, namely Thalassicolin (2,3), Aequorin (4-6), Mitrocomin (syn with Halistaurin) (7,8), Clytin (syn. with Phialidin) (8,9), Obelin (2,6,10,11), Mnemiopsin (12,13) and Berovin (12,13) have been isolated so far. All these proteins are complexes formed by an apoprotein, an imidazopyrazine chromophore (coelenterazine) and oxygen.
  • photoprotein identifies the luciferin-bound polypeptide, which is capable of luminescence, while "apophotoprotein” is used to indicate the protein without luciferin.
  • the most studied photoproteins are Aequorin, isolated from Aequorea victoria (14) and Obelin, isolated from Obelia longissima (15).
  • the photoprotein may be regenerated from the apophotoprotein by incubation with coelenterazine, molecular oxygen, EDTA and 2-mercaptoethanol or dithiothreitol. Since coelenterazine is the common luminescent substrate used by the photoproteins Aequorin, Mitrocomin, Clytin and Obelin, the light-emitting reaction is likely the same in these four photoproteins (16,17).
  • the Clytin photoprotein was cloned in 1993 by Inouye et al. (18). To date not much work has been done on this photoprotein. The primary structures of aequorin, mitrocomin, clytin and obelin were aligned and showed very strong amino acid sequence identities. The Ca 2+ -binding sites of Clytin were also found to be highly conserved (19). It was found that hydrozoan Ca 2+ -binding photoprotein differs from other Ca 2+ -binding proteins such as calmodulin and troponin C by a relatively high content of cysteine, histidine, tryptophan, proline and tyrosine residues.
  • clytin contains 198 aminoacidic residues (aa) and belongs to the family of photoproteins.
  • Photoproteins are widely used in reporter gene technology to monitor the cellular events associated with signal transduction and gene expression.
  • Photoproteins are expressed in mammalian cells to monitor calcium changes in response to different stimuli. Intracellular calcium concentrations can be measured by adding the cofactor coelenterazine to mammalian cells expressing the photoprotein and detecting photon emission, which is indicative of intracellular calcium concentration.
  • the use of cells which express both a photoprotein and a receptor involved in the modulation of intracellular calcium concentration provides a valid system for the screening of compounds for their effects on the release of intracellular calcium.
  • High throughput screening assays can also be designed using a photoprotein as reporter system. The sensitivity of the system as well as its high signal to noise ratio allow the use of small assay-volumes. Aequorin is up to now the most used photoprotein for these screening assays.
  • HTS Calcium flux assays are commonly carried out in HTS format utilizing optical screening apparatuses suited for the simultaneous analysis of a high number of samples and equipped with a luminescence imaging system with a CCD Camera detector.
  • FLIPR® Fluorometric Imaging Plate Reader, Molecular Devices Corporation, Sunnyvale, CA, USA
  • the apparatus is equipped with an optical detection device that allows for signal isolation on a cell-monolayer, thereby enhancing sensitivity for cell-based assays.
  • the excitation source can be either an Argon laser or a broadband source as a Xenon lamp.
  • the FLIPR® system With a light-tight enclosure, extremely sensitive and fast camera, and true simultaneous on-line liquid dispensing, the FLIPR® system most recent versions (FLIPR 3 and FLIPR TETRA ) have been made suitable also for luminescence assays, even if with lower sensitivity compared to CCD Camera-based equipments.
  • the invention provides an isolated photoprotein containing an amino acid sequence which:
  • the photoprotein contains an amino acid sequence which is selected from the group of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8 and 9.
  • the photoproteins of the invention show improved bioluminescence activity, higher affinity to calcium and longer-lasting light emission.
  • the Clytin sequence can be further modified without negatively affecting the photoprotein's bioluminescence activity, especially by conservative substitutions of amino acidic residues, within the indicated sequence-identity limits.
  • the Clytin sequence can be deleted of small portions, without altering its photoprotein activity.
  • the invention is directed to a polynucleotide encoding a photoprotein as defined above.
  • the polynucleotide sequences are optimized for mammalian codon usage according to SEQ ID NO: 10, 11, 12, 13, 14, 15, 16, 17.
  • the nucleic acid molecules are fused to mitochondrial target sequences (20, 21, 22).
  • the invention provides expression vectors and host cells containing the indicated polynucleotides.
  • Host cells expressing a photoprotein according to the invention produce an intense bioluminescence in response to calcium stimulation, which is much higher than that observed with natural photoproteins, in particular with the most used one, Aequorin.
  • the invention provides a cell-based assay for determining intracellular calcium concentration by means of a photoprotein according to the invention.
  • the changes in intracellular calcium concentration are determined by:
  • the assay is preferably carried out in a high-throughput format utilizing an optical screening tool or apparatus suited for multi-sample analysis, such as a luminescence imaging system with a CCD Camera detector for high and ultra high throughput applications, or with the Fluorometric Imaging Plate Reader (FLIPR® ).
  • an optical screening tool or apparatus suited for multi-sample analysis, such as a luminescence imaging system with a CCD Camera detector for high and ultra high throughput applications, or with the Fluorometric Imaging Plate Reader (FLIPR® ).
  • FLIPR® Fluorometric Imaging Plate Reader
  • cells expressing a photoprotein and a receptor involved in intracellular calcium mobilization are used to test candidate molecules for their effects on receptor modulation.
  • cells are transfected with an expression vector containing a photoprotein encoding sequence and when not endogenously present, a receptor or channel of interest.
  • the positive clones are selected and plated in a suitable medium, cultured cells are loaded with the coelenterazine substrate and the assay is started by adding the test molecule or stimulus.
  • the produced luminescence is read by a suitable detection system (CCD camera or luminometer).
  • the assay can also be run in an automatic apparatus equipped with multi-well plate reading, in particular the FLIPR® system.
  • photoprotein-expressing cells are plated in microplate wells, which, after addition of the test molecule/stimulus, are read simultaneously with signal recording.
  • High throughput screening assays set up with a photoprotein-based reporter system show improved sensitivity and signal-to-noise ratio.
  • Cells expressing a photoprotein of the invention produce an intense bioluminescence in response to calcium stimulation, which is generally higher than that observed with natural photoproteins.
  • the invention provides an assay kit containing a preparation of cells expressing an invention photoprotein under the control of a stable or inducible promoter, and reagents suitable for running the assay.
  • the photoproteins of the invention may be used as intracellular calcium indicators in diagnostic methods based on the measurement of cellular calcium ion concentration and/or cellular calcium ion influx/outflow.
  • LIC Ligation Independent Cloning
  • the codon usage of the wild-type clytin gene was adapted to the codon bias of highly expressed mammalian genes. In addition regions of very high (> 80%) or very low ( ⁇ 30%) GC content have been avoided where possible.
  • the GeneMorph II Random Mutagenesis kit (Stratagene) was used following supplier's instructions. Two different initial amounts of target DNA were used to achieve a high mutation rate, 0.1ng and 0.01ng.
  • PCR primers were appropriately designed to contain 5' LIC extensions (in italics) corresponding to sequences described in the Ek/LIC Cloning Kit (Novagen) Upper: GATGACGACGACAAG -ATGGCCGACACCGCCAG Lower: GAGGAGAAGCCCGGT -TTATCAAGGACACGAACT Amplification protocol performed in the Perkin Elmer 2400 thermocycler: 1 time the following step:
  • amplification products were analysed by electrophoresis on 1% agarose gel in 1xTAE running buffer following standard procedure, as described by Maniatis et al. The samples were compared to a DNA molecular weight marker (MWXVI, Roche).
  • the Novagen Ligation Independent Cloning (LIC) kit was used following suppliers instructions in order to obtain directional cloning of the PCR products without the need of restriction enzyme digestion or ligation reactions.
  • This strain lacks both the lon and the ompT proteases, which can. degrade proteins during purification. Hte phenotype increases the transformation efficiency (>1x108 cfu/ ⁇ g of pUC18 DNA) In addition the endA gene, that encodes endonuclease I is inactivated (no degradation of plasmid DNA).
  • Transformation efficiency was tested by using the pUC18 DNA and the pET DNA vectors and the efficiencies obtained were:
  • Transformed cells were plated on an LB agar plates and grown overnight at 37°C. After overnight colony growth, induction was obtained by adding 10 mM IPTG and 5 mM EDTA, and incubating for 4 hours at 37°C. Colonies were charged with 10 ⁇ M Coelenterazine solution and incubated overnight at 4°C in the absence of light.
  • Bioluminescence is assayed by the detection of the signal over a fixed time period of 30" at time 0, after 3 and after 5 minutes from the first measurement.
  • Restriction enzymes were purchased from New England Biolabs (Beverly, MA) and used according to supplier's instructions. Rapid DNA ligation kit and Fugene transfection reagent were purchased from Roche (Basel, CH). Coelenterazine was from Pharma Tech. International Inc. (Fairfield, NJ). All other chemicals were from standard sources and were of reagent grade or better.
  • PCR protocol was as follows: Primers: Upper primer: TCGTTGGGATCCGCCACCATGGCCGACACCGCC Lower primer: GGGCCCTCTAGATTATCAAGGCACGAA PCR reaction mix: 2 ⁇ l template 5 ⁇ l 10 x Pfx Buffer (GIBCO-LifeTechnologies) 1.5 ⁇ l 10 mM dNTPs 1 ⁇ l 50 mM MgSO 4 (GIBCO-LifeTechnologies) 2.5 ⁇ l upper primer (10 ⁇ M) 2.5 ⁇ l lower primer (10 ⁇ M) 2.5 U Platinum Pfx (GIBCO-LifeTechnologies) 35 ⁇ l H 2 O Amplification protocol performed in Perkin Elmer 2400 thermocycler: 1 time the following step: pre PCR 2' at 94°C 25 times the following steps: denaturation 15" at 94°C annealing 30" at 56°C elongation 40" at 68°C 1 time the following step: elongation 10' at 68°C Expected
  • Amplification products were analysed by electrophoresis on 1% agarose gel in IxTAE running buffer following the standard procedure, as described by Maniatis et al.
  • the PCR product was gel purified using Qiagen columns and digested with BamHI and XbaI restriction enzymes.
  • An in-house modified pcDNA3 vector (Invitrogen) has been prepared containing the sequence encoding the mitochondrial targeting peptide from subunit VIII of human cytochrome c oxidase (20, 21, 22) so that it could be used in frame at the 5' end of the codon-usage optimized photoprotein gene.
  • the amplification product obtained from the above mentioned PCR has been cloned into this modified pcDNA3 vector lacking the Neomycin resistance gene for expression in mammalian cell lines.
  • the Mutated-clytin gene is obtained from the pcrScript/hMutated-clytin vector by digestion with BamHI and XbaI.
  • the pcDNA3neo- is digested with the BamHI and XbaI restriction enzymes, and purified.
  • the Mutated-clytin gene is then ligated into the pcDNA3neo- vector to obtain pcDNA3neo-CYTO-hMutated-clytin.
  • Preculture conditions Cells were seeded for experiments when 70-80% confluent.
  • CHO cells are seeded at different cell densities in 384MTP (500, 750, 1,000, 1,500 cells/well) in growth media supplemented as above and measured with a CCD camera 24 hours and 48 hours after plating. Prior to experiments growth medium is removed and cells are loaded with Tyrode buffer plus coelenterazine at 37°C for 3 hours. Luminescence is finally monitored by CCD camera after the addition of the agonist (30 sec, kinetic).
  • Fluorometric Imaging Plate Reader (FLIPR®) Measurements
  • ATP (Sigma, A-7699) was dissolved in H 2 O at a concentration of 100 mM and stored in aliquots at -20°C. Working solution was freshly prepared in tyrode buffer.
  • Tyrode Buffer composition NaCl 130 mM, KC1 5 mM, CaCl 2 2 mM, MgCl 2 1 mM, NaHCO 3 5 mM e HEPES 20 mM, pH 7.4.
  • the Random mutant Library was obtained by using the GeneMorph II Random Mutagenesis kit (Stratagene). In order to achieve a high mutation rate two different initial amounts of target DNA were used; 0,1 ng and 0.01 ng. A total of 83305 bacterial colonies were tested for luminescence activity. Of these, 1089 were positive and therefore had bioluminescent characteristics. The best colonies were picked, for a total of 289 colonies, and of these, 16 resulted best after a re-test. Finally 8 colonies were chosen and analysed.
  • Figure 1 shows the results obtained in a three test-point CaCl 2 dose-response curve obtained with the 8 mutants.
  • Recombinant photoproteins corresponding to some mutants were produced under native conditions following a small scale purification protocol. Light emission was measured upon 1 mM calcium injection and the corresponding kinetics are shown in Fig. 4.
  • the recombinant mutant photoprotein corresponding to clone 25NO3b, was better characterized with a complete calcium dose-response curve, which can be seen in Fig. 5.
  • the recombinant photoprotein 25N03b was compared to recombinant Aequorin.
  • the light emission recorded upon 1 mM CaCl 2 injection was surprisingly higher in the case of the 25N03b mutant as shown in Fig, 6.
  • the CHOmito25N03b-expressing clone (CHOK1/mito25N03b) has been obtained by transfection of CHO-K1 cells (Materials and Methods). 48 hours after transfection the cells were trypsinized and plated into 10x96 MTP (Microtiter Plates) in complete MEM. At confluence, the 10x96 MTP were duplicated using MATRIX (Hudson, NH, USA) in 10x96 white MTP. 3 hours before measurement the medium was replaced with 50 ⁇ l/well of tyrode buffer 2 mM Ca 2+ and 10 ⁇ M coelenterazine.
  • the clones were selected on the basis of their functional response (luminescent signal) to ATP, which is known to stimulate the CHO endogenous receptor P2Y and to rise the cytoplasmic Ca 2+ concentration.
  • ATP adenosine triphosphate
  • cells were lysed by perfusion of a solution containing TritonX-100.
  • the active photoprotein was reconstituted incubating the cells with 10 ⁇ M coelenterazine diluted in tyrode buffer containing 2 mM Ca 2+ , in the dark, at 37°C, in a 5% CO 2 atmosphere for 3 hrs.
  • For light emission measurement cells were lysed in the presence of calcium and the emitted luminescence recorded.
  • the number of photons emitted during the first 30 seconds was integrated by a CCD camera and visualized on the screen. Cells transfected with an empty plasmid or untransfected did not increase photon-emission. To detect changes in calcium concentrations, 10 ⁇ M ATP was injected and the kinetics of the response determined. The curve obtained is shown in Fig. 7.
  • the final clone was seeded in 384 w/p and tested with increasing ATP concentrations as shown in the dose response curve in Fig. 8.
  • the CHOK1/mito25N03b cell line was transfected with a G-protein coupled receptor, the Adenosine A3 receptor, and with a chimeric G ⁇ protein, in order to switch the signal to the PLC/IP pathway.
  • a stable cell line was generated (from now on referred as CHOK1/mito25N03b /A3 clone).
  • the A3 receptor Upon stimulation with its agonist, the A3 receptor induces an increase in intracellular calcium concentration which is measured by mito25N03b luminescence.
  • the final clones of the CHO cell lines expressing mito25N03b and the human Adenosine A3 receptor were grown to 80-95% confluence in tissue culture flasks and harvested by trypsinization.
  • Cells were dispensed at different cell densities in 384 w/p in growth medium (DMEM/F12 containing 10% Foetal Bovine Serum) and incubated for 24 h and 48h at 37°C in a humidified incubator at 5% CO 2 .
  • the culture medium was removed and, for luminescence experiments, cells were loaded with 5 ⁇ M coelenterazine for 3h, at 37°C, 5% CO 2 .
  • Calcium response was stimulated by addition of different concentrations of ATP to each well.
  • the kinetics of flash luminescence was followed using a CCD camera, which injects reagents and records light emission from each single well.
  • the agonist and antagonist were diluted in tyrode buffer at different concentrations. Approximately 25 ⁇ l of these solutions were separately injected into each well and the response measured with the CCD camera instrumentation. The emitted light was immediately recorded at different time intervals.
  • the CHOK1/mito25N03b/A3 are tested at FLIPR 384 by measuring the fluorescence signal induced by the activation of the transfected receptor.
  • the cells are incubated with the Calcium assay kit (Molecular Devices Corporation, Sunnyvale, CA, USA).

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EP05005390A 2005-03-11 2005-03-11 Photoproteine mit erhöhter Biolumineszenz und deren Verwendung als intrazelluläre Calcium-Indikatoren Withdrawn EP1700865A1 (de)

Priority Applications (16)

Application Number Priority Date Filing Date Title
EP05005390A EP1700865A1 (de) 2005-03-11 2005-03-11 Photoproteine mit erhöhter Biolumineszenz und deren Verwendung als intrazelluläre Calcium-Indikatoren
PT06723320T PT1858921E (pt) 2005-03-11 2006-03-09 Fotoproteínas com bioluminescência melhorada e sua utilização como indicadores do cálcio intracelular
AT06723320T ATE422504T1 (de) 2005-03-11 2006-03-09 Photoproteine mit verbesserter biolumineszenz und deren verwendung als intrazelluläre kalziumindikatoren
US10/587,523 US7981602B2 (en) 2005-03-11 2006-03-09 Photoproteins with enhanced bioluminescence and assays using the same
CA002601029A CA2601029A1 (en) 2005-03-11 2006-03-09 Photoproteins with enhanced bioluminescence and their use as intracellular calcium indicators
CNA200680007800XA CN101137667A (zh) 2005-03-11 2006-03-09 生物发光增强的光蛋白及其作为细胞内钙指示剂的用途
AU2006222182A AU2006222182A1 (en) 2005-03-11 2006-03-09 Photoproteins with enhanced bioluminescence and their use as intracellular calcium indicators
EP06723320A EP1858921B1 (de) 2005-03-11 2006-03-09 Photoproteine mit verbesserter biolumineszenz und deren verwendung als intrazelluläre kalziumindikatoren
DE602006005141T DE602006005141D1 (de) 2005-03-11 2006-03-09 Photoproteine mit verbesserter biolumineszenz und deren verwendung als intrazelluläre kalziumindikatoren
RU2007133797/10A RU2407750C2 (ru) 2005-03-11 2006-03-09 Фотобелки с усиленной биолюминесценцией и их использование в качестве внутриклеточных индикаторов кальция
ES06723320T ES2321029T3 (es) 2005-03-11 2006-03-09 Fotoproteinas con bioluminiscencia potenciada y su uso como indicadores de calcio intracelulares.
PCT/EP2006/002172 WO2006094805A1 (en) 2005-03-11 2006-03-09 Photoproteins with enhanced bioluminescence and their use as intracellular calcium indicators
KR1020077021664A KR20070121689A (ko) 2005-03-11 2006-03-09 생발광이 강화된 포토프로테인 및 세포내 칼슘인디케이터로서의 이의 용도
JP2008500125A JP4962981B2 (ja) 2005-03-11 2006-03-09 強化された生物発光を示す発光タンパク質、及びその細胞内カルシウム指標としての使用
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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WO2008107104A1 (de) * 2007-03-08 2008-09-12 Bayer Schering Pharma Aktiengesellschaft Isoliertes photoprotein mtclytindecay, sowie dessen verwendung
JP2009523025A (ja) * 2006-01-11 2009-06-18 アイクスイクスアエンメ・エッセ・ピ・ア 発光幹細胞およびその使用
US11684268B2 (en) 2017-01-09 2023-06-27 Calcivis Limited Detection device

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WO2016099131A1 (ko) * 2014-12-15 2016-06-23 주식회사 엘지생활건강 피부 외용제 조성물
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995021191A1 (en) * 1994-02-04 1995-08-10 William Ward Bioluminescent indicator based upon the expression of a gene for a modified green-fluorescent protein
US5714666A (en) * 1993-02-09 1998-02-03 Children's Hospital Of Philadelphia Measurement of intracellular calcium using bioluminescent apoaequorin expressed in mammalian cells
FR2827292A1 (fr) * 2001-07-12 2003-01-17 Centre Nat Rech Scient Photoproteines mutees et leurs applications

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5360728A (en) * 1992-12-01 1994-11-01 Woods Hole Oceanographic Institution (W.H.O.I.) Modified apoaequorin having increased bioluminescent activity

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5714666A (en) * 1993-02-09 1998-02-03 Children's Hospital Of Philadelphia Measurement of intracellular calcium using bioluminescent apoaequorin expressed in mammalian cells
WO1995021191A1 (en) * 1994-02-04 1995-08-10 William Ward Bioluminescent indicator based upon the expression of a gene for a modified green-fluorescent protein
FR2827292A1 (fr) * 2001-07-12 2003-01-17 Centre Nat Rech Scient Photoproteines mutees et leurs applications

Non-Patent Citations (26)

* Cited by examiner, † Cited by third party
Title
BLINKS J R: "Use of photoproteins as intracellular calcium indicators.", ENVIRONMENTAL HEALTH PERSPECTIVES. MAR 1990, vol. 84, March 1990 (1990-03-01), pages 75 - 81, XP001205760, ISSN: 0091-6765 *
BLINKS, J ,R. ET AL.: "Measurement of Ca++ concentrations in living cells.", PROG. BIOPHYS. MOL. BIOL., vol. 40, 1982, pages 1 - 114, XP025196638, DOI: doi:10.1016/0079-6107(82)90011-6
CAMPBELL, A.K. ET AL.: "IN BIOLUMINESCENCE AND CHEMILUMINESCENCE, BASIC CHEMISTRY AND ANALYTICAL APPLICATIONS", ACADEMY PRESS, article "Hart and Herring P.J. ( 1981 ) Application of the photoprotein obelin to the mea", pages: 601 - 607
CAMPBELL, A.K.: "Extraction, partial purification and properties of obelin the calcium-activated protein from the hydroid Obelia geniculata.", BIOCHEM.J., vol. 143, 1974, pages 411 - 418
CHIESA A ET AL: "RECOMBINANT AEQUORIN AND GREEN FLUORESCENT PROTEIN AS VALUABLE TOOLS IN THE STUDY OF CELL SIGNALLING", BIOCHEMICAL JOURNAL, THE BIOCHEMICAL SOCIETY, LONDON, GB, vol. 355, no. 1, 1 April 2001 (2001-04-01), pages 1 - 12, XP009013205, ISSN: 0264-6021 *
HERRING, P.J.: "Some features of the bioluminescence of the radiolarian Thalassicola sp", MAR. BIOL., vol. 53, 1979, pages 213 - 216
ILLARIONOV B. A. ET AL.: "Sequence of the cDNA encoding the Ca++-activated photoprotein obelin from the hydroid polyp Obelia longissima.", GENE, vol. 153, no. 2, 1995, pages 273 - 4, XP004042540, DOI: doi:10.1016/0378-1119(94)00797-V
INOUYE S ET AL: "CLONING AND SEQUENCE ANALYSIS OF CDNA FOR THE CA2+-ACTIVATED PHOTOPROTEIN, CLYTIN", FEBS LETTERS, ELSEVIER SCIENCE PUBLISHERS, AMSTERDAM, NL, vol. 315, no. 3, January 1993 (1993-01-01), pages 343 - 346, XP001180448, ISSN: 0014-5793 *
INOUYE S.; TSUJI F.I.: "Cloning and sequence analysis of cDNA for the Ca(2+)-activated photoprotein, clytin", FEBS LETT., vol. 315, no. 3, 11 January 1993 (1993-01-11), pages 343 - 6, XP025593147, DOI: doi:10.1016/0014-5793(93)81191-2
JOHNSON, F.H.; SHIMOMURA, O.: "Introduction to the bioluminescence of medusae, with special reference to the photoprotein aequorin.", METHODS ENZYMOL., vol. 57, 1978, pages 271 - 291
KENDALL, J. M.; BADMINTON, M.N.: "Aequorea victoria bioluminescence moves into an exciting new era", TRENDS BIOTECHNOLOGY, vol. 16, no. 5, 1998, pages 216 - 24, XP004117786, DOI: doi:10.1016/S0167-7799(98)01184-6
LEVINE, L.D.; WARD, W.W.: "Isolation and characterization of a photoprotein ''phialidin'' and a spectrally unique green-fluorescent protein from the bioluminescent jellyfish Phialidium gregarium.", COMP. BIOCHEM. PHYSIOL, vol. 72B, 1982, pages 77 - 85, XP001039602, DOI: doi:10.1016/0305-0491(82)90013-X
MARKOVA S.V. ET AL.: "Obelin from the bioluminescent marine hydroid Obelia geniculata: cloning, expression; and comparison of some properties with those of other Ca2+-regulated photoproteins", BIOCHEMISTRY, vol. 41, no. 7, 19 February 2002 (2002-02-19), pages 2227 - 36, XP002232863, DOI: doi:10.1021/bi0117910
MORIN, J. G.; HASTINGS J. W.: "Biochemistry of the bioluminescence of colonial hydroids and other coelenterates.", J. CELL. PHYSIOL., vol. 77, 1971, pages 305 - 311
MORIN, J. G.; HASTINGS: "Energy transfer in a bioluminescent system.", J. CELL. PHYSIOL., vol. 77, 1971, pages 313 - 318
RIZZUTO, R. ET AL.: "Microdomains with high Ca2+ close to IP3-sensitive channels that are sensed by neighbouring mitochondria.", SCIENCE, vol. 262, 1993, pages 744 - 747
RIZZUTO, R. ET AL.: "Mitochondrial Ca2+ homeostasis in intact cells", J. CELL BIOL., vol. 126, 1994, pages 1183 - 1194
RIZZUTO, R. ET AL.: "Rapid changes of mitochondrial Ca2+ revealed by specifically targeted recombinant aequorin.", NATURE, vol. 358, 1992, pages 325 - 328
SHIMOMURA, O.; JOHNSON F.H.; SAIGA, Y: "Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea", J. CELL. COMP. PHYSIOL., vol. 59, 1962, pages 223 - 239
SHIMOMURA, O.; JOHNSON F.H.; SAIGA, Y: "Further data on the bioluminescent protein, aequorin", J. CELL. COMP. PHYSIOL., vol. 62, 1963, pages 1 - 8
SHIMOMURA, O.; JOHNSON, F.H.; SAIGA, Y.: "Extraction and properties of halistaurin, a bioluminescent protein from the hydromedusan Halistaura.", J. CELL. PHYSIOL., vol. 62, 1963, pages 9 - 15, XP002540540, DOI: doi:10.1002/jcp.1030620103
SHIMOMURA, O.; SHIMOMURA, A.: "Halistaurin, phialidin and modified forms of aequorin as Ca++ indicator in biological systems.", BIOCHEM. J., vol. 228, 1985, pages 745 - 749
TSUJI F I ET AL: "SITE-SPECIFIC MUTAGENESIS OF THE CALCIUM-BINDING PHOTOPROTEIN AEQUORIN", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, NATIONAL ACADEMY OF SCIENCE. WASHINGTON, US, vol. 83, no. 21, 1986, pages 8107 - 8111, XP002198115, ISSN: 0027-8424 *
TSUJI F.I. ET AL.: "Molecular evolution of the Ca(2+)-binding photoproteins of the Hydrozoa", PHOTOCHEM PHOTOBIOL. OCT, vol. 62, no. 4, 1995, pages 657 - 61
WARD, W.W.; SELIGER H.H.: "Properties of mnemiopsin, and berovin, calcium-activated photoproteins from the ctenophores Mnemiopsis sp. and Beroë ovata.", BIOCHEMISTRY, vol. 13, 1974, pages 1500 - 1510, XP002410417
WARD, W.W.; SELINGER: "Extraction and purification of calcium-activated photoprotein from the ctenophores Mnemiopsis sp. and Bern ovata.", BIOCHEMISTRY, vol. 13, 1974, pages 1491 - 1499

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JP2009523025A (ja) * 2006-01-11 2009-06-18 アイクスイクスアエンメ・エッセ・ピ・ア 発光幹細胞およびその使用
JP2009523024A (ja) * 2006-01-11 2009-06-18 アイクスイクスアエンメ・エッセ・ピ・ア 発光性トランスジェニック非ヒト動物、子孫、派生細胞（ｃｅｌｌｄｅｒｉｖａｔｉｖｅｓ）およびそれらの使用
GB2445460A (en) * 2006-12-21 2008-07-09 Lux Biotechnology Ltd Detection of tooth demineralisation
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IL185857A (en) 2012-02-29
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